Reports of a possible split emerged overnight and representatives for both stars have now told Us Magazine that the couple have decided to part.
Leoni and Duchovny married on May 6, 1997 and eventually welcomed two children, 12-year-old Madelaine and 9-year-old Kyd.
The pair briefly separated once before in late 2008, but reconciled several months later.
Reps for Duchovny and Leoni refused to speculate as to whether they will file for divorce.
Two-time Golden Globe winner Duchovny is currently starring in the Showtime drama Californication, which has been renewed for a fifth season.
Tower Heist, in which Leoni will co-star with Eddie Murphy and Ben Stiller, is released on November 4 in US and UK cinemas.
> Californication exec teases fifth season
> Meagan Good for Californication guest role